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Probes for improved melt discrimination and multiplexing in nucleic acid assays

a nucleic acid assay and detection method technology, applied in the field of molecular biology, can solve the problems of limited multiplexing capability of current real-time pcr, difficulty in quantifying the starting template, and unparallel amplification and precision capability, and achieve the effect of increasing the multiplexing capability of real-time nucleic acid amplification

Active Publication Date: 2015-02-12
LUMINEX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent is about systems and methods to amplify and detect DNA. The invention allows for the simultaneous amplification of multiple DNA sequences during real-time PCR, which increases the efficiency and accuracy of the process.

Problems solved by technology

PCR has been accepted by molecular biologists as the method of choice for nucleic acid detection because of its unparalleled amplification and precision capability.
DNA detection is typically performed at the end-point, or plateau phase of the PCR reaction, making it difficult to quantify the starting template.
However, a drawback of current real-time PCR is its limited multiplexing capability.
These requirements not only limit the practical multiplexing capability, but also increase costs since such instruments typically require multiple emission sources, detectors, and filters.

Method used

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  • Probes for improved melt discrimination and multiplexing in nucleic acid assays
  • Probes for improved melt discrimination and multiplexing in nucleic acid assays
  • Probes for improved melt discrimination and multiplexing in nucleic acid assays

Examples

Experimental program
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Effect test

example 1

Hairpin Probe Extension with Unique Melt Signatures

[0147]An extendable probe with a hairpin has been designed to provide unique melt peaks during a melt analysis to allow for greater discrimination of target identity in a melt assay, which can allow for greater multiplexing. This extendable probe can function as a probe when targeted to a sequence within an amplicon for a second level of discrimination, or it can be used as a primer in a primer set (FIGS. 1A-B).

[0148]The extendable probe comprises, in sequence from the 5′ end to the 3′ end, a reporter (star), for example, a fluorophore, a sequence capable of forming a hairpin, a modification designed to block extension (e.g., C3 spacer) during second strand synthesis, an isoG nucleotide, and a target specific sequence. During second strand synthesis using the extended probe as a template, a quencher-labeled isoC nucleotide will be incorporated opposite the isoG nucleotide (FIGS. 1C-D).

[0149]If hairpin probes are designed such that e...

example 2

Endonuclease Reaction Followed by Extension and Exonuclease Cleavage of Melt-Discrimination Probe-Template Pairs

[0152]An extendable probe is designed with a target-specific hybridization sequence at its 3′ end (Sequence B, FIG. 2A) and a melt-discrimination template-specific hybridization sequence at its 5′ end (Sequence A, FIG. 2A). In the presence of the target sequence, polymerase extension from an upstream primer during a PCR reaction cleaves the melt-discrimination template-specific hybridization sequence (FIG. 2B) and degradation of the target-specific hybridization sequence. The cleaved melt-discrimination template-specific hybridization sequence hybridizes to and extends on a melt-discrimination template (Sequence C, FIG. 2C) that has a predesigned melt-discrimination probe (Sequence D, FIG. 2C) hybridized downstream of the binding site for the melt-discrimination template-specific hybridization sequence (FIG. 2C). The 3′ end of the melt-discrimination template comprises a r...

example 3

Endonuclease Reaction Followed by Extension and Exonuclease Cleavage of Melt-Discrimination Probe-Template Pairs that Comprise an Extension Blocker

[0154]An alternative embodiment of the melt-discrimination probe-melt-discrimination template pairs described in Example 2 is presented here. In this embodiment, the melt-discrimination template (Sequence C) comprises a modification designed to block extension (e.g., C3 spacer) with the portion of the template that hybridizes to the melt-discrimination probe (Sequence D) (FIG. 3A). When Sequence A from the hydrolyzed target-specific probe hybridizes to and extends on a melt-discrimination template (Sequence C, FIG. 3A) that has a predesigned melt-discrimination probe (Sequence D, FIG. 3A) hybridized downstream of the binding site for the melt-discrimination template-specific hybridization sequence. The 3′ end of the melt-discrimination template comprises a reporter-labeled isoG nucleotide and the 5′ end of the melt-discrimination probe co...

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Abstract

Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of primers or probes that comprise a non-natural nucleotide linked to a reporter. Target nucleic acids are detected by the polymerization of a complementary probe or primer that incorporated a cognate non-natural nucleotide linked to a quencher.

Description

[0001]This application claims benefit of priority to U.S. Provisional Application Ser. No. 61 / 864,128, filed Aug. 9, 2013, the entire contents of which are hereby incorporated by reference.INCORPORATION OF SEQUENCE LISTING[0002]The sequence listing that is contained in the file named “LUMNP0119US_ST25.txt”, which is 5 KB (as measured in Microsoft Windows®) and was created on Jul. 31, 2014, is filed herewith by electronic submission and is incorporated by reference herein.BACKGROUND OF THE INVENTION[0003]1. Field of the Invention[0004]The present invention relates generally to the field of molecular biology. More particularly, it concerns the detection of nucleic acids.[0005]2. Description of Related Art[0006]Polymerase chain reaction (PCR) is a molecular biology technique for enzymatically replicating DNA without using a living organism. PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identificati...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q1/6818C12Q2525/186C12Q2527/107C12Q2561/109C12Q2525/161C12Q2525/301C12Q2533/101C12Q2537/143C12Q1/686C12Q2563/107
Inventor WHITMAN, DOUGARAB, NICOLASCOLLINS, CHUCK
Owner LUMINEX
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